| BACKGROUND AND OBJECTIVES:Gastric cancer is one of the most common gastrointestinal malignant tumors. In recent years, with comprehensive treatments used in tumors, although the mortality rate of gastric cancer has declined, there is still not a breakthrough in the treatment effects. Its morbidity and mortality remain at the forefront of various malignant tumors in our country. At present, it is generally considered that the major causes of death in patients with poor prognosis and treatment failure in gastric cancer is due to carcinoma infiltration and metastasis, and invasion and migration of cancer cells are the key links to tumor metastasis. Numerous studies have showed that epithelial-mesenchymal transition (EMT) is closely related to invasion and metastasis of malignant tumors, and it is also the research focus of tumor metastasis over the last few years. EMT is one common phenomenon during the embryogenesis developing of multicellular organisms. In EMT process, the epithelial cells’polarities disappear and change to a mesenchymal phenotype similar to interstitial fibroblast cell properties. This process involves an increase in fibroid morphology, invasiveness, resistance to apoptosis and an increase in extracellular matrix (ECM) components. EMT also enable tumor cells to acquire characteristics of stem-like cells, which means to be able to produce tumor stem cells (TSCs), and further promote tumor invasion and metastasis.High mobility group protein A2 (HMGA2) is a non-histone chromosomal protein. As an architectural transcription factor, HMGA2 itself lacks of transcriptional activity, but it may adjust other genetic transcription by altering their chromatin structures to affect embryogenesis, tissue development and tumorigenesis. HMGA2 is low or no expression in well differentiated mature tissues, but it is increased obviously in embryonic development and malignant tumors. In addition, HMGA2 may induce EMT and promote tumor cells to acquire stem cell properties via interfering with cell cycles, and maintain the differentiation and self-renewal ability of tumor cells. SOX7 is a member of the SOX F subtribe of SOX transcription factor family containing the HMG domain. Some studies have indicated that there is a transcription activation structure in the C-terminal domain of SOX7 gene. SOX7 is a negative regulator of Wnt/p-catenin signaling pathway. It may terminate cell abnormal proliferation and suppress tumor growth through inhibiting Wnt/β-catenin signaling pathway.In previous study, we used chromatin immunoprecipitation and promoter microarray (ChIP-on-chip), then analyzed ChIP through the KEGG-PATHWAY. The results showed that SOX7 might be a target gene of HMGA2 in the EMT process of gastric cancer cells by Wnt/β-catenin signaling pathway. This work aims to study how HMGA2 regulates EMT and acquisition of tumor stem cell-like properties through targets of SOX7 through gastric cancer tissues, carcinoma cells in vitro, and tumor xenografts in vivo in nude mice.METHODS:-(1) Immunohistochemistry SP method was used to detected expressions of HMGA2 and Oct4 in 158 gastric cancer specimens, associations between expression levels of HMGA2 and Oct4, relationships between HMGA2 and Oct4 immunohistochemical staining and corresponding clinicopathologic parameters, and both expressions’ influences on the prognosis of patients were analyzed.(2) To select appropriate gastric cancer cell lines (MKN-28, SGC-7901, and MKN-45), construct HMGA2 overexpression lentiviral vector, transfect MKN-28 and SGC-7901 cells to establish stable gastric cancer cell lines with overexpression of HMGA2, then verify transfection effects by western-blot.(3) Expressions of stem cell markers (CD44, Oct4) in MKN28-GFP, MKN28-A2, SGC7901-GFP, and SGC7901-A2 cell lines were examined by immunofluorescence and western-blot separately, then migration, invasion and proliferative abilities of MKN28 and SGC-7901 cells were also detected by Transwell, BrdU, and Wound Healing before and after HMGA2 overexpression.(4) Expressions of HMGA2 and SOX7 were measured by immunohistochemical staining and western-blot separately in 158 gastric cancer specimens, and associations between expression levels of both were analyzed. Meanwhile, expressions of HMGA2 and SOX7 were also detected in MKN28 and SGC7901 cell lines by western-blot before and after HMGA2 overexpression.(5) SOX7 gene promoter region was analyzed and predicted by the bioinformatics technology (UCSC software), and then construct SOX7 promoter luciferase reporter gene to further verify the relationship between HMGA2 and SOX7.(6) To design and synthesize SOX7-siRNA based on SOX7 gene promoter sequences, then transiently transfect MKN28 with endogenous high-expression of SOX7 with SOX7-siRNA, and verify transfection effects via qPCR and western-blot.(7) Expressions of EMT markers (E-cad, N-cad, and Vimentin) and stem cell markers (CD44, Oct4) were examined by western-blot before and after SOX7-siRNA interfered with MKN28 cell lines, and functions of corresponding cell lines were also detected.(8)To observe tumorigenicity, growth condition, and survival time of nude mice by models of nude mice transplantation tumors, and analyse whether EMT and tumor stem cell-like properties could be acquired in vivo after SOX-siRNA interfered with MKN28 cell lines through IHC, which could further explain whether HMGA2 could be regulated by SOX7 in the course of EMT and sternness acquisition of gastric cancer cells.RESULTS:(1) A positive correlation between HMGA2 and Oct4 expression was observed in 158 gastric cancer specimens, and both expressions were related to lymph node metastasis, vascular invasion, liver metastase, and clinical TNM stage in patients with gastric cancer.(2) Migration and invasion abilities of MKN28 and SGC-7901 cell lines were significantly enhanced, but their proliferative capacities were inhibited after MKN28 and SGC-7901 cell lines with endogenous low expression of HMGA2 were transfected by lentiviral vectors of HMGA2 overexpression. And stem cell markers CD44 and Oct4 in MKN28 and SGC-7901 cell lines were increased significantly after overexpression of HMGA2.(3) A negative correlation between HMGA2 and SOX7 expression was observed in 158 gastric cancer tissues. SOX7 promoter luciferase reporter gene showed that there is a relationship between HMGA2 and its downstream target gene SOX7, that is to say, overexpression of HMGA2 can obviously inhibit SOX7 promoter activity.(4) After SOX7-siRNA interfered with MKN28 cell lines with endogenous high-expression of SOX7, E-cadherin expression decreased, and expressions of N-cadherin, Vimentin, CD44, and Oct4 increased in MKN28 cell lines. Meanwhile, MKN28’s invasion and migration ability enhanced, but its proliferative capacity was inhibited. The subcutaneous xenograft model of nude mice indicated that the tumorigenic ability of MKN28 cells enhanced, but the growth rate slowed after MKN28 was interfered by SOX7-siRNA.CONCLUSIONS:Expression levels of HMGA2 and Oct4 were significantly higher in gastric cancer tissues compared with peritumoral tissues and normal gastric epithelium, and were also associated with invasion, metastasis, and poor prognosis in patients with gastric cancer. Stable overexpression of HMGA2 and SOX7-siRNA interfering with gastric cancer cells may promote EMT and stemness acquisition of tumor cells, and enhance invasion, migration and tumorigenic ability. A negative correlation between HMGA2 and SOX7 expressions existed in gastric cancer tissues. In addition, some binding sites might exist between HMGA2 and its downstream targets SOX7. HMGA2 can regulate EMT and sternness acquisition in gastric cancer cells by inhibiting SOX7 promoter transcription activity. |
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